沉默维生素D受体的表达对大鼠气道平滑肌细胞增殖及核因子-κB活化的影响  

Effect of VDR gene silencing on proliferation and NF-κB activation of airway smooth muscle cells

作　　者：邹淑梅 郑曦 赖国祥 余宗阳 宋颖芳 Zou Shumei;Zheng Xi;Lai Guoxiang;Yu Zongyang;Song Yingfang(Department of Pulmonary and Critical Care Medicine,900 Hospital of the Joint Logistics Team,Dong-fang Hospital,Xiamen University,Fuzhou Clinical College of Fujian Medical University,Fuzhou 350025,China)

机构地区：[1]联勤保障部队第九〇〇医院厦门大学附属东方医院福建医科大学福总临床医学院呼吸内科,福州350025

出　　处：《中华结核和呼吸杂志》2021年第6期537-542,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：福建省自然科学基金(2020J11134,2016J01474)。

摘　　要：目的研究沉默维生素D受体(VDR)基因表达对气道平滑肌细胞(ASMC)增殖能力的影响,并初步探讨核因子-κB(NF-κB)在其中的作用。方法构建特异性靶向大鼠VDR基因的RNA干扰慢病毒载体。实验分组为空白组、空载体组和干扰组。嘌呤霉素抗性筛选后建立VDR基因稳定沉默的大鼠ASMC细胞株。四甲基偶氮唑盐(MTT)法检测细胞增殖;流式细胞仪检测细胞生长周期;免疫荧光双标染色法检测NF-κB p65的核易位情况;双荧光素酶报告基因法检测NF-κB依赖的转录活性,Western blotting法检测IκBα及p-IκBα蛋白的表达水平;放线菌素D处理细胞,检测IκBαmRNA的稳定性。利用SPSS 23.0统计软件,采用多组间单因素方差(One-Way ANOVA)分析;组间两两比较采用SNK检验。结果(1)与空白组与空载体组相比,干扰组ASMC的A_(492 nm)值及G_(2)/M期细胞比例显著高(P<0.05),而G_(0/1)细胞比例明显低(P<0.05)。(2)干扰组ASMC中NF-κB亚单位p65发生明显核易位;其NF-κB报告基因的相对表达量(1.37±0.28)较空白组(1.00±0.19,P=0.031)及空载体组(0.96±0.18,P=0.027)明显高。(3)干扰组中IκBα的蛋白表达量(0.13±0.04)显著低于空白组(0.29±0.05,P=0.023)及空载体组(0.32±0.07,P=0.014)。相反,干扰组p-IκBα/IκBα为(0.86±0.04),显著高于空白组(0.41±0.07,P=0.026)及空载体组(0.37±0.05,P=0.017)。(4)干扰组ASMC的IκBαmRNA半衰期(171.31±9.67)min,较空白组[(224.69±7.95)min,P=0.032]及空载体组[(230.41±6.37)min,P=0.035]明显短。结论沉默VDR的表达能促进ASMC的细胞增殖,这一作用与其活化ASMC中的NF-κB信号通路有关。Objective To investigate the effect of VDR gene silencing on proliferation of airway smooth muscle cells(ASMCs)and elucidate the role of NF-κB.Methods A recombinant lentiviral vector specifically targeting VDR gene in rat was constructed by RNA interference.Rat ASMCs were divided into blank group,empty vector group and interference group.ASM cell line model stably silencing the VDR gene RNA expressing was selected by puromycin.Then MTT colorimetric assay and cell cycle analysis by flow cytometry were used to examine cell proliferation.The activation of nuclear factor-κB was determined by immunofluorescence double label method.Moreover,NF-κB-dependent transcription activity was tested through luciferase reporter gene assay.Western blotting was used for IκBαand phospho-IκBαprotein levels and actinomycin D treatment was used to determine IκBαmRNA stability.All statistical analyses were performed using SPSS version 23.0 software.Differences between groups were analyzed using one-way ANOVA analysis.Multiple comparisons among groups were made by Student-Newman-Keuls test.Results(1)As compared with those in the blank group and the empty vector group,the cell proliferation index(PI)and the percent of ASMCs at G_(2)/M phase in the interference group were markedly increased(P<0.05),but their percent at G_(0/1) phase was decreased(P<0.05).(2)In the interference group,the nuclear translocation of NF-κB p65 in ASMCs was obviously induced.And its level of receptor gene NF-κB p65(1.37±0.28)was significantly higher than that in the blank group(1.00±0.19,P=0.031)and in the empty vector group(0.96±0.18,P=0.027).(3)In the interference group,the IκBαprotein level in ASMCs(0.13±0.04)was obviously less than that in the blank group(0.29±0.05,P=0.023)and in the empty vector group(0.32±0.07,P=0.014).Oppositely,the p-IκBα/IκBαlevel in the interference group(0.86±0.04)was much more than that in the blank control group(0.41±0.07,P=0.026)and in the empty vector group(0.37±0.05,P=0.017).(4)In the interference group,I

关 键 词：维生素D受体 骨化三醇 肌细胞 肌 平滑 细胞增殖 核因子-κB 

分 类 号：R562.25[医药卫生—呼吸系统]